With global energy demands rising rapidly, new technologies need to be developed that utilize new resources for transportation fuels. Lignocellulosic biomass is one promising resource, where an estimated one billion tons will be available annually by 2030 in the US alone. Lignocellulosic biomass is primarily composed of plant cell wall polysaccharides, such as cellulase and hemicelluloses, which together constitute 60-70% of the biomass by weight for potential energy crops such as switchgrass. These polymers are composed of hexose and pentose sugars that can be fermented into substitutes for gasoline, diesel and jet fuel, augmenting or displacing current petroleum-based sources of liquid transportation fuels. One of the challenges of using lignocellulosic biomass for production of biofuels is the recalcitrance of plant biomass to deconstruction, a property that necessitates some form of chemical or physical pretreatment to permit enzymes or chemicals to gain access to and hydrolyze the plant polymers into fermentable sugars.
Pretreating biomass with certain classes ILs, most notably those with imidazolium-based cations, can be more efficient and tunable than other existing forms of pretreatment, and technoeconomic analysis of this route suggests that there are potential routes to economically viability. However, cellulase cocktails derived from filamentous fungi are incompatible with ILs. These enzyme cocktails can be strongly inhibited by certain ILs, necessitating expensive and inefficient washing steps to remove residual IL from the biomass prior to addition of enzymes (e.g., Li et al., Bioresource Technol 101:4900-4906, 2010; Turner et al., Green Chem 5:443-447, 2003; Park et al, PLoS One 2012, 7:e37010, 2012; Gladden et al., Appl Environ Microbiol 77:5804-5812, 2011). One solution to this issue is to develop enzyme cocktails that are tolerant to ILs. It has been shown that certain thermophilic bacterial cellulase enzymes can tolerate high levels of these ILs, and in fact these enzymes have been used to develop an IL-tolerant cellulase cocktail called JTherm (e.g., Park et al., 2012, supra; Gladden et al, 2011, supra; Datta et al., Green Chem 12:338-345, 2010; Gladden et al., Biotechnol Bioeng 109:1140-1145, 2012; Zhang et al., Green Chem 13:2083-2090, 2011. It has been further demonstrated that JTherm can be used in a one-pot IL pretreatment and saccharification bioprocessing scheme that eliminates the need to wash the pretreated biomass with water, significantly reducing the number of process steps (e.g., Shi et al., Green Chem 15:2579-2589, 2013).
Recently, complex compost-derived microbial communities were cultivated on switchgrass under thermophilic conditions to enrich for organisms that produce mixtures of IL-tolerant cellulases and xylanases (Gladden et al., 2011, supra). The community was composed of several abundant bacterial populations related to Thermus thermophilus, Rhodothermus marinus, Paenibacillus, Thermobacillus and an uncultivated lineage in the Gemmatimonadetes phylum (D'Haeseleer et al., PLoS ONE 8:e68465, 2013). The glycoside hydrolases from this community were found to have high optimum temperatures (˜80° C.) and tolerated relatively high levels of [C2mim][OAc] compared to commercial cellulase cocktails (>50% activity in the presence of 30% (v/v) [C2mim][OAc]). Therefore, these communities provide a rich reservoir of potential enzyme targets to develop thermophilic and IL tolerant cellulase cocktails. To discover the genes that encode these IL- and thermo-tolerant enzymes, metagenomic and proteomic analysis was conducted on the community (Gladden et al., 2011, supra; D'Haeseleer et al. 2013, supra)
The present invention provides IL- and thermo-tolerant cellulase enzymes, including enzymes whose activities are stimulated in the presence of ILs, which can be used in saccharification reactions to obtain sugars from lignocellulosic biomass.